Costimulation Blockade Disrupts CD4+ T Cell Memory Pathways and Uncouples Their Link to Decline in ß-Cell Function in Type 1 Diabetes.

We previously reported that costimulation blockade by abatacept limits the decline of ß-cell function and the frequency of circulating CD4+ central memory T cells (TCM) (CD45RO+CD62L+) in new-onset type 1 diabetes. In human subjects receiving placebo, we found a significant association between an increase in CD4+ TCM cells and the decline of ß-cell function. To extend and refine these findings, we examined changes in human CD4+ and CD8+ naive and memory T cell subsets at greater resolution using polychromatic flow and mass cytometry. In the placebo group, we successfully reproduced the original finding of a significant association between TCM and ß-cell function and extended this to other T cell subsets. Furthermore, we show that abatacept treatment significantly alters the frequencies of a majority of CD4+ conventional and regulatory T cell subsets; in general, Ag-naive subsets increase and Ag-experienced subsets decrease, whereas CD8+ T cell subsets are relatively resistant to drug effects, indicating a lesser reliance on CD28-mediated costimulation. Importantly, abatacept uncouples the relationship between changes in T cell subsets and ß-cell function that is a component of the natural history of the disease. Although these data suggest immunological markers for predicting change in ß-cell function in type 1 diabetes, the finding that abatacept blunts this relationship renders the biomarkers nonpredictive for this type of therapy. In sum, our findings point to a novel mechanism of action for this successful immunotherapy that may guide other disease-modifying approaches for type 1 diabetes.

Peripheral immune circadian variation, synchronisation and possible dysrhythmia in established type 1 diabetes.

AIMS/HYPOTHESIS: The circadian clock influences both diabetes and immunity. Our goal in this study was to characterise more thoroughly the circadian patterns of immune cell populations and cytokines that are particularly relevant to the immune pathology of type 1 diabetes and thus fill in a current gap in our understanding of this disease. METHODS: Ten individuals with established type 1 diabetes (mean disease duration 11 years, age 18-40 years, six female) participated in a circadian sampling protocol, each providing six blood samples over a 24 h period. RESULTS: Daily ranges of population frequencies were sometimes large and possibly clinically significant. Several immune populations, such as dendritic cells, CD4 and CD8 T cells and their effector memory subpopulations, CD4 regulatory T cells, B cells and cytokine IL-6, exhibited statistically significant circadian rhythmicity. In a comparison with historical healthy control individuals, but using shipped samples, we observed that participants with type 1 diabetes had statistically significant phase shifts occurring in the time of peak occurrence of B cells (+4.8 h), CD4 and CD8 T cells (~ +5 h) and their naive and effector memory subsets (~ +3.3 to +4.5 h), and regulatory T cells (+4.1 h). An independent streptozotocin murine experiment confirmed the phase shifting of CD8 T cells and suggests that circadian dysrhythmia in type 1 diabetes might be an effect and not a cause of the disease. CONCLUSIONS/INTERPRETATION: Future efforts investigating this newly described aspect of type 1 diabetes in human participants are warranted. Peripheral immune populations should be measured near the same time of day in order to reduce circadian-related variation.

Efficacy of GAD-alum immunotherapy associated with HLA-DR3-DQ2 in recently diagnosed type 1 diabetes.

AIMS/HYPOTHESIS: The aim of this study was to determine if retention of C-peptide following immunotherapy using recombinant GAD65 conjugated to aluminium hydroxide (GAD-alum) is influenced by HLA risk haplotypes DR3-DQ2 and DR4-DQ8. METHODS: HLA-dependent treatment effect of GAD-alum therapy on C-peptide retention in individuals with recent-onset type 1 diabetes was evaluated using individual-level patient data from three placebo-controlled, randomised clinical trials using a mixed repeated measures model. RESULTS: A significant and dose-dependent effect was observed in individuals positive for the genotypes that include HLA-DR3-DQ2 but not HLA-DR4-DQ8 and in the broader subgroup of individuals positive for all genotypes that include HLA-DR3-DQ2 (i.e. including those also positive for HLA-DR4-DQ8). Higher doses (three or four injections) showed a treatment effect ratio of 1.596 (95% CI 1.132, 2.249; adjusted p = 0.0035) and 1.441 (95% CI 1.188, 1.749; adjusted p = 0.0007) vs placebo for the two respective HLA subgroups. CONCLUSIONS/INTERPRETATION: GAD65-specific immunotherapy has a significant effect on C-peptide retention in individuals with recent-onset type 1 diabetes who have the DR3-DQ2 haplotype. Graphical abstract.

GAD-alum immunotherapy in type 1 diabetes expands bifunctional Th1/Th2 autoreactive CD4 T cells.

AIMS/HYPOTHESIS: Antigen-specific therapy aims to modify inflammatory T cell responses in type 1 diabetes and restore immune tolerance. One strategy employs GAD65 conjugated to aluminium hydroxide (GAD-alum) to take advantage of the T helper (Th)2-biasing adjuvant properties of alum and thereby regulate pathological Th1 autoimmunity. We explored the cellular and molecular mechanism of GAD-alum action in the setting of a previously reported randomised placebo-controlled clinical trial conducted by Type 1 Diabetes TrialNet. METHODS: In the clinical trial conducted by Type 1 Diabetes TrialNet, participants were immunised with 20 µg GAD-alum (twice or three times) or alum alone and peripheral blood mononuclear cell samples were banked at baseline and post treatment. In the present study, GAD-specific T cell responses were measured in these samples and GAD-specific T cell lines and clones were generated, which were then further characterised. RESULTS: At day 91 post immunisation, we detected GAD-specific IL-13+ CD4 T cell responses significantly more frequently in participants immunised with GAD-alum (71% and 94% treated twice or three times, respectively) compared with those immunised with alum alone (38%; p = 0.003 and p = 0.0002, respectively) accompanied by high secreted levels of IL-13, IL-4 and IL-5, confirming a GAD-specific, GAD-alum-induced Th2 response. Of note, GAD-specific, IL-13+ CD4 T cells observed after immunisation co-secreted IFN-γ, displaying a bifunctional Th1/Th2 phenotype. Single-cell transcriptome analysis identified IL13 and IFNG expression in concert with the canonical Th2 and Th1 transcription factor genes GATA3 and TBX21, respectively. T cell receptor ß-chain (TCRB) CDR3 regions of GAD-specific bifunctional T cells were identified in circulating naive and central memory CD4 T cell pools of non-immunised participants with new-onset type 1 diabetes and healthy individuals, suggesting the potential for bifunctional responses to be generated de novo by GAD-alum immunisation or via expansion from an existing public repertoire. CONCLUSIONS/INTERPRETATION: GAD-alum immunisation activates and propagates GAD-specific CD4 T cells with a distinctive bifunctional phenotype, the functional analysis of which might be important in understanding therapeutic responses.

GAD vaccine reduces insulin loss in recently diagnosed type 1 diabetes: findings from a Bayesian meta-analysis.

AIMS/HYPOTHESIS: GAD is a major target of the autoimmune response that occurs in type 1 diabetes mellitus. Randomised controlled clinical trials of a GAD + alum vaccine in human participants have so far given conflicting results. METHODS: In this study, we sought to see whether a clearer answer to the question of whether GAD65 has an effect on C-peptide could be reached by combining individual-level data from the randomised controlled trials using Bayesian meta-analysis to estimate the probability of a positive biological effect (a reduction in C-peptide loss compared with placebo approximately 1 year after the GAD vaccine). RESULTS: We estimate that there is a 98% probability that 20 µg GAD with alum administered twice yields a positive biological effect. The effect is probably a 15-20% reduction in the loss of C-peptide at approximately 1 year after treatment. This translates to an annual expected loss of between -0.250 and -0.235 pmol/ml in treated patients compared with an expected 2 h AUC loss of -0.294 pmol/ml at 1 year for untreated newly diagnosed patients. CONCLUSIONS/INTERPRETATION: The biological effect of this vaccination should be developed further in order to reach clinically desirable reductions in insulin loss in patients recently diagnosed with type 1 diabetes.

Statistical considerations when analyzing biomarker data.

Biomarkers have become, and will continue to become, increasingly important to clinical immunology research. Yet, biomarkers often present new problems and raise new statistical and study design issues to scientists working in clinical immunology. In this paper I discuss statistical considerations related to the important biomarker problems of: 1) The design and analysis of clinical studies which seek to determine whether changes from baseline in a biomarker are associated with changes in a metabolic outcome; 2) The conditions that are required for a biomarker to be considered a "surrogate"; 3) Considerations that arise when analyzing whether or not a predictive biomarker could act as a surrogate endpoint; 4) Biomarker timing relative to the clinical endpoint; 5) The problem of analyzing studies that measure many biomarkers from few subjects; and, 6) The use of statistical models when analyzing biomarker data arising from count data.

Heterogeneity in recent-onset type 1 diabetes - a clinical trial perspective.

BACKGROUND: Type 1 diabetes (T1D) TrialNet is a National Institutes of Health-sponsored clinical trial network aimed at altering the disease course of T1D. The purpose of this study is to evaluate age-dependent heterogeneity in clinical, metabolic and immunologic characteristics of individuals with recent-onset T1D, to identify cohorts of interest and to aid in planning of future studies. METHODS: Eight hundred eighty-three individuals with recent-onset T1D involved in five TrialNet studies were categorized by age as follows: ≥18 years, 12-17 years, 8-12 years and <8 years. Data were compared with healthy age-matched subjects in the National Health and Nutrition Examination Survey. RESULTS: Only 2.0% of the individuals overall were excluded from trial participation because of insufficient C-peptide values (<0.2 pmol/mL). A disproportionate number of these subjects were <8 years old. Leukopenia was present in 21.2% of individuals and lymphopenia in 11.6%; these frequencies were markedly higher than age-matched healthy National Health and Nutrition Examination Survey population. Of the cohort, 24.5% were overweight or obese. Neither high-risk human leukocyte antigen type DR3 nor DR4 was present in 31% of adults and 21% of children. CONCLUSIONS: The ability of recent-onset T1D patients to meet key entry criteria for TrialNet studies, including C-peptide >0.2 pmol/mL, varies by age. Lower C-peptide level requirements for younger participants and other aspects of heterogeneity of recent-onset T1D patients, such as white blood cell count abnormalities and body mass index should be considered in the design of future clinical studies.

Disease progression among 446 children with newly diagnosed type 1 diabetes located in Scandinavia, Europe, and North America during the last 27 yr.

OBJECTIVE: To clarify whether the rate of decline in stimulated C-peptide (SCP) from 2 to 15 months after diagnosis has changed over an interval of 27 yr. RESEARCH DESIGN AND METHODS: The rate of decline in SCP levels at 1, 2, 3, 6, 9, 12, and 15 months after diagnosis was compared in four paediatric cohorts from Scandinavian and European countries including 446 children with new onset type 1 diabetes (T1D, 1982-2004). Findings were evaluated against 78 children (2004-2009) from the TrialNet studies. RESULTS: The mean rate of decline [%/month (±SEM)] in SCP for a 10-yr-old child was 7.7%/month (±1.5) in the 1982-1985 Cohort, 6.3%/month (±1.7) in the 1995-1998 Cohort, 7.8%/month (±0.7) in the 1999-2000 Cohort, and 10.7%/month (±0.9) in the latest 2004-2005 Cohort (p = 0.05). Including the TrialNet Cohort with a rate of decline in SCP of 10.0%/month (±0.9) the differences between the cohorts are still significant (p = 0.039). The rate of decline in SCP was negatively associated with age (p < 0.0001), insulin antibodies (IA) (p = 0.003), and glutamic acid decarboxylase-65 (GAD65A) (p = 0.03) initially with no statistically significant effect of body mass index (BMI) Z-score at 3 months. Also, at 3 months the time around partial remission, the effect of age on SCP was significantly greater in children ≤5 yr compared with older children (p ≤ 0.0001). CONCLUSIONS: During the past 27 yr, initial C-peptide as well as the rate of C-peptide decline seem to have increased. The rate of decline was affected significantly by age, GAD65A, and IA, but not BMI Z-score or initial C-peptide.

The effects of ambient lighting in chest radiology reading rooms.

Under typical dark chest radiography reading room conditions, a radiologist's pupils contract and dilate as their visual focus intermittently shifts between the high luminance monitor and the darker background wall, resulting in increased visual fatigue and degradation of diagnostic performance. A controlled increase of ambient lighting may minimize these visual adjustments and potentially improve comfort and accuracy. This study was designed to determine the effect of a controlled increase of ambient lighting on chest radiologist nodule detection performance. Four chest radiologists read 100 radiographs (50 normal and 50 containing a subtle nodule) under low (E=1 lx) and elevated (E=50 lx) ambient lighting levels on a DICOM-calibrated, medical-grade liquid crystal display. Radiologists were asked to identify nodule locations and rate their detection confidence. A receiver operating characteristic (ROC) analysis of radiologist results was performed and area under ROC curve (AUC) values calculated for each ambient lighting level. Additionally, radiologist selection times under both illuminance conditions were determined. Average AUC values did not significantly differ (p>0.05) between ambient lighting levels (estimated mean difference=-0.03; 95% CI, (-0.08, 0.03)). Average selection times decreased or remained constant with increased illuminance. The most considerable decreases occurred for false positive identification times (35.4±18.8 to 26.2±14.9 s) and true positive identification times (29.7±18.3 to 24.5±15.5 s). No performance differences were statistically significant. Study findings suggest that a controlled increase of ambient lighting within darkly lit chest radiology reading rooms, to a level more suitable for performance of common radiological tasks, does not appear to have a statistically significant effect on nodule detection performance.

Equity and Prevention of Cardiovascular Diseases in Latin America and the Caribbean.

Non-communicable diseases, particularly cardiovascular diseases, are the leading cause of decreased life expectancy and death in Latin America and the Caribbean. Although a lifestyle, which includes no tobacco use, good nutrition, and regular physical activity is touted as key to health, the environmental, racial, social and economic conditions, which underpin lifestyle are often ignored or considered only secondarily. Placing the main responsibility on a patient to change their lifestyle or to simply comply with pharmacological treatment ignores the specific conditions in which the individual lives. Furthermore, there are major disparities in access to both healthy living conditions as well as access to medical care. There is sufficient evidence to support advocating for policies that support healthy living, particularly healthy food choices. Progress is being made to improve the food environment with enactment of front of package nutritional labels. However, policies were enacted only after intense regional research and advocacy supporting their implementation. Government officials must rise above the pressures of commercial interests and support health-promoting policies or be exposed as self-interest groups themselves. Strong advocacy is required to persuade officials that all policies should take health into consideration both to improve lives and economies.

Immune and Metabolic Effects of Antigen-Specific Immunotherapy Using Multiple ß-Cell Peptides in Type 1 Diabetes.

Type 1 diabetes is characterized by a loss of tolerance to pancreatic ß-cell autoantigens and defects in regulatory T-cell (Treg) function. In preclinical models, immunotherapy with MHC-selective, autoantigenic peptides restores immune tolerance, prevents diabetes, and shows greater potency when multiple peptides are used. To translate this strategy into the clinical setting, we administered a mixture of six HLA-DRB1*0401-selective, ß-cell peptides intradermally to patients with recent-onset type 1 diabetes possessing this genotype in a randomized placebo-controlled study at monthly doses of 10, 100, and 500 µg for 24 weeks. Stimulated C-peptide (measuring insulin functional reserve) had declined in all placebo subjects at 24 weeks but was maintained at ≥100% baseline levels in one-half of the treated group. Treatment was accompanied by significant changes in islet-specific immune responses and a dose-dependent increase in Treg expression of the canonical transcription factor FOXP3 and changes in Treg gene expression. In this first-in-human study, multiple-peptide immunotherapy shows promise as a strategy to correct immune regulatory defects fundamental to the pathobiology of autoimmune diabetes.

Hospital certification for optimizing cardiovascular disease and stroke quality of care and outcomes.

Cardiovascular disease and stroke remain leading causes of mortality, disability, and rising healthcare expenditures in the United States. Although a number of organizations provide hospital accreditation, recognition, and certification programs, existing programs do not address cardiovascular disease and stroke care in a comprehensive way. Current evidence suggests mixed findings for correlation between accreditation, recognition, and certification programs and hospitals' actual quality of care and outcomes. This advisory discusses potential opportunities to develop and enhance hospital certification programs for cardiovascular disease and stroke. The American Heart Association/American Stroke Association is uniquely positioned as a patient-centered, respected, transparent healthcare organization to help drive improvements in care and outcomes for patients hospitalized with cardiovascular disease and stroke. As a part of its commitment to promoting high-quality, evidence-based care for cardiovascular and stroke patients, it is recommended that the American Heart Association/American Stroke Association explore hospital certification programs to develop truly meaningful programs to facilitate improvements in and recognition for cardiovascular disease and stroke quality of care and outcomes. Future strategies should standardize objective, unbiased assessments of hospital structural, process, and outcome performance while allowing flexibility as technology and methodology advances occur.

Prostate cancer outcome and tissue levels of metal ions.

BACKGROUND: There are several studies examining prostate cancer and exposure to cadmium, iron, selenium, and zinc. Less data are available on the possible influence of these metal ions on prostate cancer outcome. This study measured levels of these ions in prostatectomy samples in order to examine possible associations between metal concentrations and disease outcome. METHODS: We obtained formalin fixed paraffin embedded tissue blocks of prostatectomy samples of 40 patients with PSA recurrence, matched 1:1 (for year of surgery, race, age, Gleason grading, and pathology TNM classification) with tissue blocks from 40 patients without recurrence (n = 80). Case-control pairs were compared for the levels of metals in areas adjacent to tumors. Inductively coupled plasma-mass spectrometry (ICP-MS) was used for quantification of Cd, Fe, Zn, and Se. RESULTS: Patients with biochemical (PSA) recurrence of disease had 12% lower median iron (95 µg/g vs. 111 µg/g; P = 0.04) and 21% lower zinc (279 µg/g vs. 346 µg/g; P = 0.04) concentrations in the normal-appearing tissue immediately adjacent to cancer areas. Differences in cadmium (0.489 µg/g vs. 0.439 µg/g; 4% higher) and selenium (1.68 µg/g vs. 1.58 µg/g; 5% higher) levels were not statistically significant in recurrence cases, when compared to non-recurrences (P = 0.40 and 0.21, respectively). CONCLUSIONS: There is an association between low zinc and low iron prostate tissue levels and biochemical recurrence in prostate cancer. Whether these novel findings are a cause or effect of more aggressive tumors, or whether low zinc and iron prostatic levels raise implications for therapy, remains to be investigated.

Pioglitazone versus glimepiride on coronary artery calcium progression in patients with type 2 diabetes mellitus: a secondary end point of the CHICAGO study.

OBJECTIVE: To compare coronary artery calcium (CAC) progression between 2 treatment groups, pioglitazone versus glimepiride. METHODS AND RESULTS: The CHICAGO (Carotid Intima-Media Thickness in Atherosclerosis Using Pioglitazone) study demonstrated that pioglitazone significantly decreased carotid intima-media thickness progression compared with glimepiride in patients with type 2 diabetes mellitus. The CAC level was determined at baseline and at the end of 72 weeks of treatment in the pioglitazone (n=146) and glimepiride (n=153) treatment groups using electron beam computed tomography. There was no difference in CAC progression between the treatment groups. By using backward and forward selection models, age, race/ethnicity, and baseline apolipoprotein B level predicted CAC progression. There was no relationship between carotid intima-media thickness and CAC progression during the study. CONCLUSIONS: There was no difference in CAC progression in patients with type 2 diabetes mellitus treated with pioglitazone or glimepiride. Age, race/ethnicity, and baseline apolipoprotein B level predicted CAC progression in patients with type 2 diabetes mellitus.

Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans.

The study of regulatory networks in human pathogens such as Cryptococcus neoformans provides insights into host-pathogen interactions that may allow for correlation of gene expression patterns with clinical outcomes. In the present study, deletion of the cryptococcal copper-dependent transcription factor 1 (Cuf1) led to defects in growth and virulence factor expression in low copper conditions. In mouse models, cuf1Delta strains exhibited reduced dissemination to the brain, but no change in lung growth, suggesting copper is limiting in neurologic infections. To examine this further, a biologic probe of available copper was constructed using the cryptococcal CUF1-dependent copper transporter, CTR4. Fungal cells demonstrated high CTR4 expression levels after phagocytosis by macrophage-like J774.16 cells and during infection of mouse brains, but not lungs, consistent with limited copper availability during neurologic infection. This was extended to human brain infections by demonstrating CTR4 expression during C. neoformans infection of an AIDS patient. Moreover, high CTR4 expression by cryptococcal strains from 24 solid organ transplant patients was associated with dissemination to the CNS. Our results suggest that copper acquisition plays a central role in fungal pathogenesis during neurologic infection and that measurement of stable traits such as CTR4 expression may be useful for risk stratification of individuals with cryptococcosis.

Making the diagnosis of acute appendicitis: do more preoperative CT scans mean fewer negative appendectomies? A 10-year study.

PURPOSE: To determine the frequency of preoperative computed tomography (CT) in the evaluation of patients suspected of having appendicitis at one institution during the past 10 years and to determine whether changes in CT utilization were associated with changes in the negative appendectomy rate. MATERIALS AND METHODS: Institutional review board approval was obtained, and a waiver of informed consent was granted for this HIPAA-compliant study. A surgical database search yielded medical record numbers of 925 patients (526 [ 56.9%] men and 399 [43.1%] women; mean age, 38 years (range, 18-95 years]) who underwent urgent appendectomy between January 1998 and September 2007. Patients who were younger than 18 years of age at the time of surgery were excluded. CT, pathology, and surgery reports were reviewed. By using logistic regression, changes in the proportion of patients undergoing CT and in the proportion of patients undergoing each year appendectomy in which the appendix was healthy were evaluated. Subgroup analyses based on patient age ( 45 years) and sex also were performed. RESULTS: Prior to urgent appendectomy, 18.5% of patients underwent preoperative CT in 1998 compared with 93.2% of patients in 2007. The negative appendectomy rate for women 45 years of age and younger decreased from 42.9% in 1998% to 7.1% in 2007. However, the timing of the decline in negative appendectomy rates for women 45 years and younger could not be proved to be associated with the increase in CT use. There was no significant trend toward a lower negative appendectomy rate for men regardless of age or for women older than 45 years of age with increased use of preoperative CT. The shift from single-detector CT to multidetector CT and the use of decreasing section thickness also correlated with a reduction in false-positive diagnoses. CONCLUSION: Rising utilization of preoperative CT and advances in technology coincided with a decrease in the negative appendectomy rate for women 45 years and younger but not in men of any age or women older than 45 years.